Morchella esculenta Polysaccharide Alleviates Lipopolysaccharide-Induced Experimental Hepatitis in Mice

Abstract

Objective

This study aimed to evaluate the potential protective effects of Morchella esculenta polysaccharide (MEP) on lipopolysaccharide (LPS)–induced experimental hepatitis in mice.

Methods

Fifty Kunming mice were randomly assigned to five groups: control, LPS, and MEP treatment groups receiving high (800 mg/kg), medium (400 mg/kg), or low (200 mg/kg) doses. MEP was administered intragastrically for 21 consecutive days. From day 14, mice in the LPS and MEP-treated groups received intraperitoneal injections of LPS (4.0 mg/kg) for seven consecutive days, while control mice received normal saline. Serum liver function–related biochemical indicators, as well as levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), were measured. Histopathological changes were evaluated by hematoxylin and eosin staining. Hepatic expression of pigment epithelium-derived factor (PEDF) and patatin-like phospholipase domain-containing 2 (PNPLA2) was assessed by immunohistochemical staining.

Results

LPS exposure resulted in evident hepatic injury, characterized by altered biochemical parameters, elevated inflammatory cytokine levels, and pronounced histopathological damage. MEP-treated mice exhibited higher serum albumin levels and lower levels of total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyltransferase, total bile acid, TNF-α, IL-1β, and the AST/ALT ratio. Histological examination further demonstrated attenuation of hepatic pathological damage in MEP-treated mice. In addition, MEP administration was associated with increased hepatic immunoreactivity of PEDF and PNPLA2.

Conclusion

MEP administration ameliorated LPS-induced experimental hepatitis in mice and was associated with improved liver biochemical indicators, reduced inflammatory cytokine levels, and increased hepatic PEDF and PNPLA2 expression, suggesting its potential hepatoprotective effects in inflammatory liver injury.

Introduction

The liver is the largest digestive gland and plays a pivotal role in detoxification and metabolism in animals. Its normal morphology and physiological function are essential for maintaining systemic homeostasis. Acute hepatitis is characterized by acute onset and rapid progression and is typically attributed to infections or toxic factors, which may confer a high risk of disseminated intravascular coagulation in animals. ¹ Additionally, acute hepatitis can induce severe and potentially fatal liver injury and may further deteriorate into cirrhosis, liver failure, and even liver cancer if not effectively treated. ² At present, the prognosis of severe acute hepatitis remains poor, as the disease lacks specific therapeutic drugs and is primarily managed by supportive therapy to allow sufficient time for hepatic recovery. ¹ Therefore, the development of effective pharmacological interventions for acute hepatitis is urgently needed.

Morchella esculenta polysaccharide (MEP), commonly present in the form of glycoprotein complexes or dextran-like macromolecules, ³ exhibits a wide range of pharmacological activities, including anti-oxidative, anti-aging, anti-tumor, anti-fatigue, immunomodulatory, hepatoprotective, and hypolipidemic effects.^4,5 In recent years, MEP has attracted increasing research attention. Pigment epithelium-derived factor (PEDF), a multifunctional soluble glycoprotein, belongs to the serine protease inhibitor superfamily and was originally isolated from the culture medium of fetal retinal pigment epithelial cells. PEDF exerts anti-inflammatory, anti-oxidative stress, anti-apoptotic, anti-angiogenic, anti-tumor, and neurotrophic effects by interacting with specific receptors on the cell membrane. ⁶ Patatin-like phospholipase domain containing 2 (PNPLA2) is a rate-limiting lipolytic enzyme encoded by the ATGL/PNPLA2 gene that catalyzes the initial step of triglyceride hydrolysis. PNPLA2 is implicated in multiple physiological and pathological processes, including chronic inflammation, lipid mobilization, and obesity. A prior study revealed that ATGL knockout led to excessive lipid accumulation in mouse liver tissue. ⁷ Accordingly, this study analyzed the effects and potential mechanisms of MEP on acute hepatitis using a lipopolysaccharide (LPS)-induced experimental hepatitis model in mice, with the aims of providing experimental evidence for the development of MEP-related health products and therapeutic strategies for hepatitis.

Materials and Methods

Ethical Approval and Statement of Human and Animal Rights

All animal procedures in this study were reviewed and approved by the Experimental Animal Ethics Committee of Longnan Normal University (Approval No. 2024-0010) and the Institutional Animal Care and Use Committee of the School of Agriculture and Forestry Technology (Approval No. 2024-0011). All experiments were conducted in strict accordance with the approved protocols and relevant institutional guidelines for the care and use of laboratory animals.

Animals and Main Reagents

Specific pathogen-free Kunming mice (8 weeks old, body weight 22−28 g, equal numbers of males and females) were purchased from the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences [SCXK (Gan) 2020-0002]. MEP (DG3156; purity ≥ 98%) was purchased from Huilin Biotechnology Co., Ltd (Xi'an, China). Enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor (TNF)-α and interleukin (IL)-1β, endogenous biotin-blocking kits, and SP hypersensitivity immunohistochemical kits were obtained from Solarbio (Beijing, China). Primary rabbit polyclonal antibodies against PEDF and PNPLA2 were provided by Asiapeptide Biotechnology Center (Nanjing, China). 3,3’-Diaminobenzidine tetrahydrochloride (DAB) was acquired from Saipei Biotechnology Co., Ltd (Wuhan, China). Isoflurane was obtained from RWD Life Science (Shenzhen, China), and 24-parameter biochemical reagent strips were provided by MNCHIP (Tianjin, China).

Main Instruments

The main instruments used in this study included a microtome (RM2125 RTS; Leica, Wetzlar, Germany), a DM500 microscopic imaging system (Leica), a Celercare V5 automatic biochemical analyzer for animals (MNCHIP), an ELISA microplate reader (Multiskan FC; Thermo Fisher Scientific (China) Co., Ltd, Shanghai, China), and an FA1204 electronic analytical balance (Lichen Instrument Technology Co., Ltd, Shanghai, China).

Animal Modeling and Grouping

Mice were randomly allocated into five groups: control, LPS, LPS + high-dose MEP (HMEP), LPS + medium-dose MEP (MMEP), and LPS + low-dose MEP (LMEP) groups (10 mice per group). Mice in the LPS and control groups were intragastrically administered normal saline for 21 consecutive days, whereas those in three MEP-treated groups were gavaged with MEP at doses of 800, 400, and 200 mg/kg, respectively, over the same period. From day 14, mice in the LPS and MEP-treated groups were intraperitoneally injected with LPS (4.0 mg/kg) under isoflurane-assisted anesthesia for 7 consecutive days. Control mice received intraperitoneal injections of an equivalent volume of normal saline via the same procedure. All outcome parameters were assessed after completion of model establishment.

Examination of IL-1β, TNF-α, and Liver Function–Related Biochemical Indicators

Twenty-four hours following the final LPS challenge, mice were euthanized under deep isoflurane anesthesia. Blood specimens obtained from the orbital venous plexus were allowed to coagulate in separator tubes, followed by cold centrifugation (4000 rpm, 10 min, 4 °C). The resulting serum fraction was harvested for further measurements.

Serum liver function–related biochemical indicators, including albumin (ALB), total bilirubin (TBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), and total bile acid (TBA), were measured using commercially available biochemical assay reagent strips on a Celercare V5 automatic biochemical analyzer for animals (MNCHIP, Tianjin, China), according to the manufacturer's protocols and established enzymatic colorimetric methods, as previously described.^8,9

Serum levels of IL-1β and TNF-α were determined using commercially available ELISA kits (Solarbio) in accordance with the manufacturers’ protocols. ¹⁰

Tissue Sampling and Immunohistochemical Staining

Mice were euthanized under deep anesthesia induced by isoflurane inhalation 24 h after the final intraperitoneal injection of LPS. Liver tissues were promptly excised and immersed in 4% paraformaldehyde for fixation at 4°C. After fixation, samples were thoroughly rinsed with running tap water for 12 h, processed routinely, and embedded in paraffin, as previously described by Chen et al Paraffin blocks were sectioned into 4-μm slices for subsequent histological and immunohistochemical analyses. ¹¹ For histopathological examination, sections were subjected to hematoxylin and eosin(HE) staining to observe the hepatic morphological characteristics. In detail, sections were deparaffinized in xylene, rehydrated through graded ethanol solutions, stained with hematoxylin, differentiated and blued with tap water, followed by eosin counterstaining, alcohol dehydration, xylene clearing, and mounting with neutral gum. Immunohistochemical staining was adopted to analyze the localization of PNPLA2 and PEDF, with positive immunoreactivity appearing as yellow-brown to dark-brown staining. After deparaffinization and rehydration, antigen retrieval was carried out, followed by SP-based immunohistochemical staining as per the manufacturer's instructions. Tissue sections were sequentially incubated in a humidified chamber with reagent A (endogenous peroxidase blocking solution), reagent B (normal goat serum), reagent C (biotin-conjugated goat anti-rabbit IgG), and reagent D (streptavidin-horseradish peroxidase) for 15, 15, 20, and 20 min, respectively. Subsequently, following incubation with reagent B, sections were exposed to rabbit polyclonal primary antibodies against PNPLA2 and PEDF (1:200 dilution) for overnight incubation at 4°C. After completion of reagent D treatment, diaminobenzidine (DAB) substrate was applied for chromogenic development for 5 min. Between each incubation step, the sections were gently rinsed three times with 0.01 mol/L phosphate-buffered saline for 5 min per wash. Finally, the sections were subjected to hematoxylin counterstaining, followed by dehydration using graded ethanol, xylene clarification, resin mounting, and subsequent microscopic observation and image acquisition.

Image and Data Analysis

Following immunohistochemical staining, tissue sections were examined and photographed under a microscope. Ten representative sections were selected, and five random high-power fields (400×) were captured from each section. Image-Pro Plus 6.0 software was employed for quantitative analysis. Prior to measurement, optical density (OD) calibration was performed. Integrated OD (IOD) and corresponding target areas were obtained using the color segmentation and separation functions. Regions of interest were manually delineated via the Count/Size module with the Select Colors tool. The mean OD of PNPLA2- and PEDF-positive staining was subsequently calculated using the formula: mean OD = IOD/positively stained area).

Statistical Analysis

The experimental data were statistically analyzed with SPSS software (version 20.0). Quantitative data were presented as mean ± standard deviation. Intergroup comparisons were performed using one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) post hoc test for pairwise comparisons. A two-tailed P value < 0.05 was considered statistically significant, and P < 0.01 indicated a stronger level of statistical significance.

Results

Effects of MEP on serum Biochemical Indicators in LPS-Induced Experimental Hepatitisin Mice

Serum biochemical changes following MEP administration in mice subjected to LPS-induced experimental hepatitis are illustrated in Figure 1. Compared with the control group, LPS administration resulted in a significant decrease in serum ALB levels, accompanied by marked increases in TBIL, AST, ALT, ALP, GGT, TBA, and the AST/ALT ratio (all P < 0.01), confirming successful induction of hepatic injury.

Figure 1.

Changes in serum biochemical indicators in LPS-induced mice after MEP treatment. Note: * P < 0.05, ** P < 0.01 compared to the control group; # P < 0.05, ## P < 0.01 compared to the LPS group; Δ P < 0.05 for comparisons among the three MEP-treated groups. The same notation applies to the following figures.

Relative to the LPS group, MEP treatment produced significant improvements in multiple serum biochemical parameters. In the LPS + HMEP group, serum ALT, TBA, and the AST/ALT ratio were markedly reduced (P < 0.01), accompanied by a significant decline in ALP levels (P < 0.05). In the LPS + MMEP group, significant decreases were observed in ALP and GGT (P < 0.01), together with reductions in TBIL, AST, TBA, and the AST/ALT ratio (P < 0.05). In the LPS + LMEP group, significant reductions were observed in serum ALT and ALP (P < 0.01), along with a moderate decrease in AST levels (P < 0.05) compared with the LPS group.

Further comparison among MEP-treated groups revealed that serum ALT levels were remarkably lower in the LPS + HMEP group than in the LPS + MMEP group (P < 0.05), while serum GGT levels were significantly lower in the LPS + MMEP group than in the LPS + HMEP group (P < 0.05). No significant differences were observed in the remaining parameters among MEP-treated groups.

Effects of MEP on serum TNF-α and IL-1β Levels in Mice with LPS-Induced Experimental Hepatitis

The effects of MEP on serum inflammatory cytokine levels in LPS-induced experimental hepatitis were evaluated (Figure 2). Compared with the control group, LPS administration resulted in significantly elevated serum levels of IL-1β and TNF-α (P < 0.01), indicating successful induction of a systemic inflammatory response.

Figure 2.

Changes in serum TNF-α and IL-1β levels in LPS-induced mice after MEP treatment.

Compared with the LPS group, MEP treatment partially attenuated LPS-induced cytokine elevation. In particular, serum TNF-α levels were significantly reduced in the LPS + HMEP group (P < 0.05). No significant differences were detected in serum IL-1β or TNF-α levels between the LPS group and the other MEP-treated groups.

Effect of MEP on Hepatic Histopathological Alterations in LPS-Induced Experimental Hepatitis in Mice

HE staining revealed that livers from the control mice exhibited preserved lobular architecture, with clearly identifiable hepatocytes, central veins, interlobular bile ducts, hepatic sinusoids, and hepatic cords. Hepatocytes displayed a typical polygonal morphology, containing one to two centrally located nuclei with visible nucleoli, and were arranged in well-organized hepatic plates. Hepatic sinusoids were located between hepatic cords, and interlobular bile ducts were found in the portal areas (Figure 3A). In the LPS group, hepatic architecture was markedly disrupted. Hepatocytes appeared swollen with indistinct cell borders, accompanied by pale cytoplasm containing multiple round vacuoles of different sizes. Hepatic sinusoids were compressed and narrowed due to cellular swelling (Figure 3B). Compared with the LPS group, the LPS + HMEP group displayed relatively preserved hepatic architecture with more clearly defined lobular structures (Figure 3C). In the LPS + MMEP group, partial restoration of hepatic architecture was observed; however, some hepatic cords remained indistinct and scattered cytoplasmic vacuoles were still present (Figure 3D). In the LPS + LMEP group, hepatocytes showed blurred cellular boundaries with persistent cytoplasmic vacuolation, accompanied by incomplete reconstruction of hepatic sinusoids and hepatic cords (Figure 3E).

Figure 3.

He staining of mouse livers (400×). Note: A. control group; B. LPS group; C. LPS + HMEP group; D. LPS + MMEP group; E. LPS + LMEP group. a. hepatocytes; b. cytoplasm; c. nucleus; d. hepatic sinusoids; e. hepatic cords; f. central veins; g. vascular endothelial cells; h. interlobular bile ducts.

Immunohistochemical Staining Results of PEDF in Mouse Livers

As depicted in Figure 4, there were light purple cytoplasmic staining, dark purple nuclear staining, and a pale blue background, with no other staining in the blank control group (Figure 4F). In the experimental groups, hepatocytes demonstrated tan to yellow-brown cytoplasmic staining, while nuclei and non-cellular structures were counterstained purple, demonstrating absence of nonspecific background staining in the immunohistochemical staining results of PEDF. PEDF-positive immunoreactivity was observed in hepatocytes of all experimental groups, with varying staining intensities. In control mice, PEDFimmunoreactivity was moderate and evenly distributed throughout the hepatic parenchyma. In LPS-challenged mice, PEDF staining intensity was heterogeneous, with reduced or absent immunoreactivity observed in scattered hepatocytes. Among the MEP-treated groups, the LPS + MMEP group showed the most pronounced PEDF positivity, characterized by strong brown cytoplasmic staining.

Figure 4.

Immunohistochemical staining of PEDF in mouse livers (400×). Note: A. control group; B. LPS group; C. LPS + HMEP group; D. LPS + MMEP group; E. LPS + LMEP group; F. blank control group. a. hepatocytes; b. cytoplasm; c. nucleus; d. hepatic sinusoids; e. hepatic cords; f. central veins; g. vascular endothelial cells; h. interlobular bile ducts.

Effects of MEP on PEDF Expression in the Livers of LPS-Induced Experimental Hepatitis Mice

In mouse livers, PEDF expression was markedly lower in the LPS and LPS + LMEP groups, whereas substantially higher in the LPS + MMEP group than in the control group (P < 0.05). Versus the LPS group, hepatic expression was remarkably elevated in the LPS + MMEP group (P < 0.01). No significant differences were found among the remaining groups (Figure 5).

Immunohistochemical Staining Results of PNPLA2 in Mouse Livers

According to immunohistochemical staining results of PNPLA2 in mouse livers (Figure 6), liver sections from the blank control group exhibited light purple cytoplasmic staining, dark purple nuclear counterstaining, and a pale blue background, with no specific PNPLA2 immunoreactivity detected (Figure 6F). In the experimental groups, hepatocytes demonstrated tan to yellow-brown cytoplasmic staining, while nuclei were counterstained purple, indicating the absence of nonspecific background staining in the immunohistochemical SP staining results of PNPLA2. PNPLA2 immunoreactivity was observed in hepatocytes of all groups, with variable staining intensity. Among the MEP-treated groups, the LPS + HMEP group exhibited the strongest PNPLA2 positivity, followed by the LPS + LMEP group. PNPLA2-positive staining was limited to focal areas within the hepatocyte cytoplasm in the LPS + MMEP group.

Figure 5.

Changes in PEDF expression in the livers of LPS-induced mice after MEP treatment detected by immunohistochemistry.

Figure 6.

Immunohistochemical staining of PNPLA2 in mouse livers (400×). Note: A. control group; B. LPS group; C. LPS + HMEP group; D. LPS + MMEP group; E. LPS + LMEP group; F. blank control group. a. hepatocytes; b. cytoplasm; c. nucleus; d. hepatic sinusoids; e. hepatic cords; f. central veins; g. vascular endothelial cells; h. interlobular bile ducts.

Figure 7.

Changes in PNPLA2 expression in the livers of LPS-induced mice after MEP treatment measured by immunohistochemistry.

Effects of MEP on PNPLA2 Expression in the Livers of LPS-Induced Experimental Hepatitis Mice

PNPLA2 expression in mouse livers was substantially elevated in the LPS + HMEP group as compared to both the control (P < 0.05) and LPS (P < 0.01) groups. No significant differences in PNPLA2 expression was observed among the remaining groups (Figure 7).

Discussion

Various inflammatory agents, including dextran sodium sulfate (DSS) and LPS, have been widely used to establish experimental models of inflammatory diseases. For example, Chen et al ¹² successfully established a murine model of ulcerative colitis using 2.5% DSS. LPS, a major component of the outer membrane of Gram-negative bacteria, is a potent inducer of inflammatory responses through the stimulation of pro-inflammatory cytokine production. Previous studies have demonstrated that LPS treatment elicits robust inflammatory responses both in vitro and in vivo, including in RAW264.7 macrophages and various rodent models of tissue injury.^13,14 Consistent with these observations, the present study demonstrated that repeated intraperitoneal injection of 4 mg/kg LPS for seven consecutive days was sufficient to induce experimental hepatitis in mice, as evidenced by abnormal liver biochemical parameters, elevated serum IL-1β and TNF-α levels, and pronounced histopathological alterations in liver tissues.

LPS-induced inflammatory responses are known to involve multiple intracellular signaling pathways, including mitogen-activated protein kinase (MAPK) cascades. MAPKs are serine/threonine kinases widely expressed in eukaryotic cells and are mainly classified into c-Jun N-terminal kinases (JNK), extracellular signal–regulated kinases (ERK), and p38 MAPK. ¹⁵ Among these, p38 MAPK has been extensively implicated in inflammatory regulation and has been reported to modulate transcription factors such as ATF-2 and NF-κB, which are involved in the transcriptional control of pro-inflammatory cytokines including IL-1β and TNF-α.^16,17 In the present study, LPS administration markedly increased serum IL-1β and TNF-α levels, both of which were significantly reduced following MEP treatment.^18,19 However, MAPK activation and related phosphorylation events were not directly assessed in this study. Therefore, the involvement of MAPK-related signaling is discussed here based on previously published literature rather than direct experimental evidence. The observed suppression of inflammatory cytokines suggests that MEP may be associated with attenuation of LPS-induced inflammatory responses, while the specific signaling pathways involved remain to be clarified in future studies.

Previous studies have reported that polysaccharides isolated from Morchella esculenta are primarily composed of monosaccharides such as glucose, galactose, and mannose, with β-(1→3) and β-(1→6) glycosidic linkages forming branched polysaccharide structures. Such structural characteristics are commonly observed in mushroom-derived polysaccharides and are considered important determinants of their biological activities, including anti-inflammatory and immunomodulatory effects. Although the detailed structural features of MEP were not characterized in the present study, these reported polysaccharide structures provide an important biochemical context for interpreting the observed biological effects.^20,21

PEDF is a multifunctional endogenous protein involved in lipid metabolism, inflammation, and cellular homeostasis. Both clinical and experimental studies have documented altered PEDF expression in metabolic disorders, including obesity and fatty liver disease, highlighting its role in lipid mobilization and triglyceride metabolism.^22,23 PNPLA2, also known as adipose triglyceride lipase, is a key enzyme responsible for triglyceride hydrolysis and lipid droplet turnover in the liver and other tissues. Notably, PNPLA2 deficiency has been shown to exacerbate liver inflammation and increase mortality following LPS challenge. ²⁴ Emerging evidence suggests that dysregulated lipid and fatty-acid metabolism plays a critical role in amplifying inflammatory responses across a spectrum of inflammatory diseases. For example, alterations in fatty acid metabolism driven by intestinal dysbiosis have been shown to exacerbate psoriasis-like inflammatory phenotypes, highlighting the close interplay between metabolic reprograming and inflammation. In this context, the modulation of PEDF- and PNPLA2-related lipid metabolic factors observed in the present study may be relevant to the inflammatory processes involved in LPS-induced experimental hepatitis.^25,26 In the present study, LPS administration was associated with reduced hepatic expression of PEDF and PNPLA2, whereas MEP treatment was accompanied by increased expression of both proteins. Notably, unlike previous studies that primarily focused on PEDF alone or examined polysaccharide effects in metabolic or non-inflammatory disease models, the present study emphasizes the associations between MEP treatment and concurrent modulation of PEDF and PNPLA2 within an LPS-induced acute inflammatory hepatitis model. This integrative perspective links inflammatory liver injury with lipid metabolism–related regulatory factors under inflammatory stress conditions. These findings indicate an association between MEP treatment and modulation of PEDF- and PNPLA2-related lipid metabolic pathways under inflammatory conditions, without establishing a direct causal relationship.

Limitations and Future Perspectives

Several limitations of the present study should be acknowledged. First, the observed changes in PEDF and PNPLA2 expression provide correlative evidence, whereas functional validation experiments, such as genetic knockdown or pharmacological inhibition of these targets, were not performed to establish causal relationships. Second, the absence of a MEP-only treatment group represents a limitation of the current experimental design, as it precludes evaluation of the basal effects of MEP in healthy animals. Third, key inflammatory signaling pathways, including MAPK activation and downstream phosphorylation events, were not directly assessed. Future studies will incorporate appropriate control groups together with molecular signaling analyses and comprehensive cytokine profiling to further elucidate the mechanisms underlying the hepatoprotective effects of MEP.

Conclusion

In summary, this study demonstrates that MEP confers a protective effect against LPS-induced inflammatory liver injury in mice. Rather than focusing solely on individual experimental readouts, the collective findings highlight the capacity of this natural polysaccharide to improve hepatic inflammatory status and maintain liver homeostasis under acute inflammatory stress. The observed modulation of in liver injury–related biochemical parameters, inflammatory cytokines, and lipid metabolism–associated factors suggests that MEP may represent a promising candidate for further investigation as a hepatoprotective agent in inflammatory liver disorders. In particular, future investigations will aim to directly link the observed changes in PEDF and PNPLA2 with specific inflammatory signaling pathways and cytokine responses, thereby providing mechanistic insight into the hepatoprotective effects of MEP.